This proposal is directed to solving the problem of the slow rate of generation of high resolution structures of integral membrane proteins that can be useful as drug targets and in medicine. The group combines established expertise in membrane protein structural biology, which has led to 16 structures deposited in the Protein Data Bank, as well as cutting-edge advances in methods development. Experience has shown that the paucity of high resolution membrane protein structure is a consequence of several recognizable bottlenecks in the structure determination pipeline, including: (i) co-expression of the subunits of hetero-subunit prokaryotic proteins;(ii) expression of eukaryotic membrane proteins;(iii) determination of stability and functionality;(iv) recognition of (a) the role of lipid in the structure and in the rate of crystallization and (b) proteolytic degradation that can preclude purification of active protein. A suite of new expression approaches will be developed targeting (i) and (ii), with complementary analysis approaches in (ii). Problems (i-iv) lead to uncertainties in the formation of well-diffracting crystals, which can take weeks or months to generate. Early detection of crystal growth would greatly decrease the time required for crystal screening and allow access to a greater phase-space of crystallization conditions. We describe an innovative methodology, SONICC (second order nonlinear optical imaging of chiral crystals), for sensitive and selective detection of incipient protein crystals as small as 100 nm, prepared in screening platforms with volumes as low as 0.5 picoliter. Its ability to distinguish membrane protein crystals of the maltose transporter and cytochrome b6f complex has been confirmed. This approach will greatly speed generation of diffraction-quality 3-dimensional and 2-dimensional crystals, generated both in surfo and in meso. A range of integral membrane protein targets has been selected, with an emphasis on ABC and hetero-oligomeric proteins;(i) ABC: maltose and ribose transporters, ABCBl, ABCG2, and Sur-Kir6.2;(ii) non-ABC: twin-arginine translocase;Kdp-ATPase;NADH dehydrogenase. PUBLIC HEALTH RELEVANCE: Integral membrane proteins control all traffic between cell compartments, assembly of the membranes themselves, the supply of nutrients to the cell, its energization, and communication of the cell with the extracellular environment. The atomic structures of membrane proteins is crucial for an understanding of the molecular basis of human diseases and the development of drugs to combat them or ameliorate their consequences.